This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have used SSRL beam time on this proposal to conduct crystallographic studies on enzymes responsible for the biosynthesis of two tRNA modifications: queosine (Q) and threonyl carbamoyladenosine (t6A). These are two modifications of the tRNA anticodon loop that alter codon definitions and fine tune ribosome function during translation. Q biosynthesis is an established drug target for anti-shigellosis antibiotics. Similarly, due to its requirement for HIV replication, t6A modification of host-cell tRNA has been proposed as a potential target for the development of anti-HIV pharmaceuticals. Using a combination of comparative genomic, biochemical, and structural methods, we identified enzymes involved in the biosynthesis of Q and t6A. Using SSRL x-rays, we determined the structure of GCYH-IB, our newly discovered and first enzyme in the Q biosynthesis pathway in prokaryotes, in complex with a substrate analog and we collected native and heavy-atom derivative data for YkvM, the enzyme that catalyzes the third step in Q biosynthesis. We also determined the crystal structure of YrdC, a universally conserved and essential enzyme involved in t6A biosynthesis, in complex with its ATP substrate. In this renewal, we plan to start crystallographic studies of GCYH-IBinhibitor complexes and of QueA, which catalyzes a late step in Q biosynthesis, in complex with its tRNA substrate. We currently have crystals of GCYH-IB-ligand complexes and crystallization of the latter complex is underway. We plan to continue to use the SSRL beamline automation technology and remote access for these studies.